In the past, a large number of pharmacologists and chemists made efforts to chemically alter and/or modify the in vivo activity of naturally occurring, physiologically active molecules. These efforts associated with physiologically active substances were focused mainly on increasing specific in vivo activity, prolonging in vivo activity, reducing toxicity, eliminating or reducing side effects, or modifying specific physiological activities. When a physiologically active substance is chemically modified, it loses some or most of its physiological activities in many cases. However, in some cases, the modification could result in an increase or change in physiological activity. In this regard, many studies have been focused on chemical modification capable of achieving desired physiological activity, and most studies have involved covalently bonding a physiologically active substance (drug) to a physiologically acceptable carrier.
To stabilize proteins and prevent enzymatic degradation and clearance by the kidney, a polymer having high solubility, such as polyethylene glycol (hereinafter, referred to simply as “PEG”), was conventionally used to chemically modify the surface of a protein drug. Since PEG binds in a non-specific manner to a specific region or various regions of a target protein, it has the effects of increasing protein solubility, stabilizing the protein and preventing protein hydrolysis, and has no specific side effects (Sada et al., J. Fermentation Bioengineering 71: 137-139, 1991). However, despite capability to enhance protein stability, this PEG coupling has problems of greatly reducing titers of physiologically active proteins and reducing yield due to PEG's reactivity with proteins decreasing with increasing molecular weight of PEG.
Recently, polymer-protein drug conjugates have been suggested. For example, as described in U.S. Pat. No. 5,738,846, a conjugate can be prepared by linking an identical protein drug to both ends of PEG to improve the activity of the protein drug. Also, as described in International Pat. Publication No. WO 92/16221, two different protein drugs can be linked to both ends of PEG to provide a conjugate having two different activities. However, these methods are not effective in sustaining the activity of protein drugs.
Kinstler et al. reported that a fusion protein prepared by coupling granulocyte-colony stimulating factor (G-CSF) to human albumin has improved stability (Kinstler et al., Pharmaceutical Research 12(12): 1883-1888, 1995). However, in this publication, since the modified drug, having a G-CSF-PEG-albumin structure, showed a only about four-fold increase in residence time in the body and a slight increase in serum half-life compared to the single administration of the native G-CSF, it has not been industrialized as an effective long-acting formulation for protein drugs.
An alternative method for improving in vivo stability of physiologically active proteins includes linking a physiologically active protein gene to a gene encoding a protein having high serum stability by genetic recombination and culturing of an animal cell transfected with the recombinant gene to produce a fusion protein. For example, a fusion protein can be prepared by conjugating albumin, known to be most effective in enhancing protein stability, or its fragment to a physiologically active protein of interest by genetic recombination (International Pat. Publication Nos. WO 93/15199 and WO 93/15200, European Pat. Publication No. 413,622). A fusion protein of interferon-alpha and albumin, developed by the Human Genome Science Company and marketed under the trade name ALBUFERON™, has a half-life increased from 5 hours to 93 hours in monkeys, but is problematic in terms of having a greatly decreased in vivo activity less than 5% compared to unmodified interferon-alpha (Osborn et al., J. Phar. Exp. Ther. 303(2): 540-548, 2002).
Recombinant DNA technologies were applied to fuse a protein drug to an immunoglobulin Fc fragment. For example, interferon (Korean Pat. Laid-open Publication No. 2003-9464), and interleukin-4 receptor, interleukin-7 receptor or erythropoietin (EPO) receptor (Korean Pat. Registration No. 249572) were previously expressed in mammals in a form fused to an immunoglobulin Fc fragment. International Pat. Publication No. WO 01/03737 describes a fusion protein comprising a cytokine or growth factor linked to an immunoglobulin Fc fragment through peptide linkage. In addition, U.S. Pat. No. 5,116,964 discloses proteins fused to the amino- or carboxyl-terminal end of an immunoglobulin Fc fragment by genetic recombination. U.S. Pat. No. 5,349,053 discloses a fusion protein comprising IL-2 fused to an immunoglobulin Fc fragment through peptide linkage. Other examples of Fc fusion proteins prepared by genetic recombination include a fusion protein of interferon-beta or its derivative and an immunoglobulin Fc fragment (International Pat. Publication NO. WO 0/23472), and a fusion protein of IL-5 receptor and an immunoglobulin Fc fragment (U.S. Pat. No. 5,712,121), a fusion protein of interferon alpha and the Fc region of immunoglobulin G4 (U.S. Pat. No. 5,723,125), and a fusion protein of CD4 protein and the Fc region of immunoglobulin G2 (U.S. Pat. No. 6,451,313).
However, these Fc fusion proteins, in which a polypeptide/protein is linked to the N- or C-terminal end of an Fc fragment through peptide linkage, are problematic as follows. Recombinant production of an Fc fusion protein can be achieved only by expression of a nucleic acid molecule encoding the Fc fusion protein in a single polypeptide/protein form in a single host cell. Thus, since the entire fusion protein is glycosylated or aglycosylated by this system, fusion is impossible between glycosylated and aglycosylated proteins. Also, these Fc fusion proteins mediate effector functions by the Fc region. Through the effector functions of the Fc region, they fix complements or bind to cells expressing FcRs, leading to lysis of specific cells, and induce the production and secretion of several cytokines inducing inflammation, leading to unwanted inflammation (U.S. Pat. No. 6,656,728; Zheng et al., J. Immunology, 1999, 163:4041-4048; Huang et al., Immunology letters, 2002, 81:49-58). Further, the fusion creates a new amino acid sequence, not present in humans, at a connection region between the Fc region and the protein partner, which could potentially induce immune responses in humans.
Many efforts have been made to prepare an immunoglobulin or immunoglobulin fragment retaining a long serum half-life but being deficient in effector functions. Cole et al. reported that, when amino acid residues of the CH2 region at positions 234, 235 and 237, known to play an important role in binding to Fc receptors, are replaced with alanine to produce an Fc derivative having a reduced binding affinity to Fc receptors, the ADCC activity is inhibited (Cole et al., J. Immunol. 159: 3613-3621, 1997). Also, U.S. Pat. No. 5,605,690 discloses a TNFR-IgG1 Fc fusion protein which is prepared by genetic recombination using an IgG1 Fc fragment having amino acid alterations in the complement binding region or receptor binding region of immunoglobulin Fc. However, conspicuous improvement was not achieved by any of these variants. For example, Fc may have increased immunogenicity compared to the native human Fc region due to the presence of unsuitable amino acid residues and may lose preferable Fc functions.
On the other hand, pegylation of immunoglobulins forming antigen-antibody complexes has been introduced, for example, for oral administration (J. Immunological Methods, 1992, 152:177-190) or to prevent the induction of complement reaction by aggregation (Biochimica et Biophysica Acta, 1984, 788:248-255). U.S. Pat. No. 4,732,863 employed a pegylation method in order to reduce immunogenicity of monoclonal antibodies and decrease non-specific binding of the antibodies to Fc receptors. However, such pegylation is carried out in a non-specific modification fashion using PEG having a molecular weight of 1 to 5 kDa to pegylate the entire immunoglobulin. Thus, these pegylation methods are disadvantageous in terms of having difficulty in retaining the Fab functions and controlling the degree of pegylation.
In addition, a site-selective pegylation method was reported, which comprises primary protection via coupling to a ligand, and then pegylation. U.S. Pat. No. 6,548,644 employed such a pegylation method to inhibit the immunogenicity, enhance the solubility and increase the serum half-life of a TNFR-Fc fusion protein. The fusion protein is protected using TNF as a protecting agent and then pegylated, thereby pegylating only sites not participating in ligand binding. When 20% of lysine residues were pegylated using PEG having a molecular weight of 1 to 5 kDa, Fc receptor binding was inhibited. However, this pegylation method has drawbacks as follows: FcRn binding sites can be pegylated, leading to a reduction in serum half-life; protection and deprotection steps are very complicated; and a homogeneous pegylated product is difficult to obtain.
As described above, reported PEG-modification methods are focused on the removal of immunogenicity or inhibition of non-specific Fc receptor binding of therapeutic immunoglobulin or Fc fusion proteins. However, there is no attempt at describing modification of a native or recombinant immunoglobulin Fc fragment by a pegylation method for use as a carrier.
Prior to the present invention, the present inventors found that, when an Fc fragment, which is not the entire immunoglobulin but a peptide fragment, is linked with a drug in a non-fused protein form, it improves the in vivo duration of action of the drug and minimizes a reduction in the in vivo activity of the drug, and submitted patent applications for use of the Fc fragment as a carrier and application thereof (Korean Pat. Application Nos. 10-2004-092780, -092781, -092782 and -092783; International Pat. Application Nos. PCT/KR2004/002942, 002943, 002944 and 002945; submitted on Nov. 13, 2004).
The present inventors found that, when the Fc fragment useful as a carrier is pegylated and used as a carrier, it does not have increased sensitivity to proteolytic enzymes, retains its binding capacity to FcRn but is deficient in binding to FcR I, II, III and C1q, and has a serum half-life similar to that of native Fc.